Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Abstract

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH 2 -terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K i value of 6.2 ؎ 0.55 nM. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.Proteases play key roles in diverse processes regulating plant growth, development, and responses to environmental stimuli. They are necessary for protein turnover, strict protein quality control, and degrading specific sets of proteins. Comparative genomics analyses could provide valuable insights into the abundance and roles of various plant protease families. For example, the Arabidopsis thaliana genome has over 550 protease sequences corresponding to almost 3% of the proteome, representing all five catalytic types: serine, cysteine, aspartic acid, metallo, and threonine (1, 2). Of these, serine proteases appear to be the largest class of plant proteases, although protease activity has been demonstrated only by a few of them.Cucumisin (EC 3.4.21.25) is an extracellular thermostable alkaline serine protease that is expressed at high levels in melon fruits (Cucumis melo L.). It comprises more than 10% of the total juice protein and is synthesized in the central parts of the fruits (3). Cucumisin is synthesized and accumulated only in melon fruits, and a cis-regulatory enhancer element in the cucumisin promoter regulates fruit-specific expression of the cucumisin gene (4). We have determined the complete nucleotide sequence of a cucumisin cDNA, the first sequenced plant serine protease, and found that cucumisin is a member of the subtilisin (EC 3.4.21.62) superfamily characterized by a catalytic triad of three amino acids: Asp, His, and Ser (5). The primary structure of cucumisin deduced from the cDNA sequence revealed that it is synthesized as a precursor consisting of four functional domains: a possible signal peptide (22 amino acid residues); NH 2 -terminal prosequence (88 residues); 54-kDa protease domain (505 residues), which is the active enzyme domain of the 67-kDa native cucumisin; and 14-kDa COOHterminal polypeptide (116 residues), which arises by limited autolysis of the 67-kDa native cucumisin (3, 5). The optimal...

Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease

“…As previously reported for the PP of cucumisin (30), SBT3PP was found to be insoluble in E. coli and accumulated in inclusion bodies. The recombinant protein was solubilized in 8 M urea and purified to apparent homogeneity by affinity chromatography on nickel-agarose beads under denaturing conditions ( Fig.…”

“…The data indicate that weak secondary structure is present in the free SBT3PP. SBT3PP thus resembles the PP of cucumisin with respect to secondary structure content and stability (30). In contrast, PPs of mammalian PCs are only marginally stable on their own (with the exception of the PP in PC1 from Mus musculus), and the PPs of bacterial subtilisins are fully unfolded at 25°C (35,37,38).…”

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

“…Inset shows a sketch of the C-terminal flexible region with hydrophobic residues highlighted. (31,32). In this case the C-terminal junction point lies directly within the active site and inhibits activity.…”

Identification of Interaction Site of Propeptide toward Mature Carboxypeptidase Y (mCPY) Based on the Similarity between Propeptide and CPY Inhibitor (IC)

“…16,17) In a study of serine protease subtilisins and their propeptides, the functions of subtilisin propeptides as intramolecular chaperons and inhibitors were identified, [18][19][20] and the structure of the subtilisinpropeptide complex was also determined. 20) Structural analysis indicated that the C-terminal strand of the propeptide binds to the subtilisin substrate-binding cleft.…”